1. Field of the Invention
The present invention relates to a dry immunoassay element in which a homogeneous enzyme immunoassay is utilized. More particularly, the present invention relates to an immunoassay element comprising a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of a labelling enzyme and a reagent layer containing a fragmenting enzyme for further fragmenting the diffusible material into lower molecular weight product, wherein the non-diffusible substrate is a substrate which selectively reacts with the labelling enzyme and avoids reacting with the fragmenting enzyme.
2. Description of the Related Art
Analyses of the constituents originated from the living body or chemicals contained in the body fluids, such as blood and urine, are useful for diagnosing the condition of diseases or judging the course of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one method for analyzing such constituents (ligands) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into heterogeneous systems for which B/F (Bound/Free) separation must be effected, and homogeneous system for which B/F separation is not necessary. The reactions in the homogeneous system are based on the phenomenon that the enzymatic activity of the labeling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. It is considered that the enzymatic activity is suppressed by a steric hindrance caused by binding the enzyme to the substrate or a change in three-dimensional structure of the enzyme, when the antibody which is generally a large molecule is bound to the antigen in the enzyme-labelled antigen.
When the antigen is a high polymer, suppression of enzymatic activity by the antigen-antibody binding reaction may be detected by labelling the antibody with an enzyme.
Meanwhile, in the routine clinical tests in which a number of test samples are to be handled, it is demanded that the individual samples should be analyzed by simple operations, more desirously by automated operation sequence.
To comply with the demand, dry analysis elements have been proposed (see, for example, Unexamined Japanese Patent Publication Nos. 53888/1974 (Corresponding to U.S. Pat. No. 3,992,158), 90859/1980 (corresponding U.S. Pat. No. 4,258,001), 164356/1980 (U.S. Pat. No. 4,292,272), 222769/1985 (EP 0162302A), 77356/1984 (corresponding to EP 0097952A), 102388/1984 and 501866/1986 (U.S. Pat. No. 4,459,358.)
A dry analysis element has been known, in which an enzyme-labelled antibody is utilized and reacted in a homogeneous enzyme immunological reaction (see Unexamined Japanese Patent Publication No. 321360/1989 which corresponds to EP 0347839A). This known dry analysis element comprises the following three reagent ingredients in the same or different layers in the composite multi-layered structure:                (A) An antigen having a high molecular weight (a coupling product of a ligand or a derivative thereof with a high molecular weight compound; hereinafter referred to as “polymerized antigen”);        (B) A water-insoluble high polymer substrate; and        (C) A conjugate of an antibody against the ligand and an enzyme for the substrate.        
The antigen supplied by spotting onto the analysis element binds to the antibody-enzyme conjugate through a competitive reaction with the reaction of the polymerized antigen. The complex of antigen-antibody-enzyme reacts with the water-insoluble high polymer substrate to form a soluble lower molecular weight product. On the other hand, the complex of polymerized antigen and enzyme-labelled antibody formed by the binding with the polymerized antigen cannot exhibit the enzymatic activity to the high polymer substrate. Accordingly, as the quantity of the antigen in the sample is increased, the product produced by the enzymatic reaction increases. This product is allowed to diffuse into a detection layer where the quantity of the product is determined by measuring the optical density of an absorption resulted by the colored chemical group, to make it possible to analyze the antigen in the sample quantitatively.
The immunoassay element disclosed in Japanese Patent No. 2576910 (corresponding to U.S. Pat. No. 5,569,589 and EP 0451848A) is an improvement of the aforementioned immunoassay element. This immunoassay element has a reagent layer containing a fragmenting enzyme for further fragmenting the decomposition product by the reaction of labelling enzyme, so that the fragmented lower molecular weight product is detected for further sensitization of the element.
When the analyte or ligand is a macromolecular antigen, the immunoassay element described in the specification of Japanese Patent No. 2576913 (corresponding to U.S. patent application Ser. No. 07/763,198) may be used. This prior art element has the following two components either in a same layer or in different layers:
(A) Water-insoluble high polymer substrate; and
(B) Conjugate of an antibody to the macromolecular antigen and an enzyme for the substrate.
Likewise to the immunoassay element described in the specification of Japanese Patent No. 2576910 (U.S. Pat. No. 5,569,589) a reagent layer containing a fragmenting enzyme for further fragmenting the decomposition product by the action of labelling enzyme is provided so that the fragmented product having a lower molecular weight is detected to improve the sensitivity.
In any event, the non-diffusible substrate will not migrate into the reagent layer. Further, it has been believed that the fragmenting enzyme will never migrate backward from the reagent layer to the substrate layer which overlies it. It has been heretofore customary, therefore, to deny that the substrate specificity of a non-diffusible substrate is very important and therefore to select any non-diffusible substrate which can react at all with both fragmenting enzyme and labelling enzyme.
An assay element on which a sample solution has applied, however, suffers diffusion of a soluble component, if only to a slight extent, from the lower reagent layer to the upper substrate layer. The present inventors' study of this diffusion has revealed that very small amount of the fragmenting enzyme migrates from the reagent layer to the substrate layer on the upstream side and reacts with the non-diffusible substrate to form a low molecular weight product, and that this product constitutes itself a noise hardly deserving to be ignored. Heretofore, such noise has had the possibility of degrading the accuracy of assay.
Further, the macromolecular substrate retained as a non-diffusible substrate in the substrate layer happens to contain a substrate of a low polymerization degree or a substrate of an unduly small particle diameter as an extraneous component or impurities, in a minute amount. Some of this macromolecular substrate inevitably migrates from the upper substrate layer to the lower reagent layer in consequence of the advance of supply of the sample solution. The macromolecular substrate thus migrated in a minute amount is destined to react with the fragmenting enzyme in the reagent layer and the product of this reaction is also fated to form a cause of noise.
The present inventors, thereupon, have prepared an immunoassay element by using as a non-diffusible substrate such a substrate as reacts solely with a labelling enzyme and avoids reacting with a fragmenting enzyme and then studied this immunoassay element to determine the quality thereof, to find that this immunoassay element is superior to the conventional immunoassay element not only in sensitivity but also in reproducibility and durability of aging (storage stability).